S-Nitrosoglutathione induces functional ΔF508-CFTR in airway epithelial cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl− channel in airway epithelial cells, plays an important role in maintaining the volume of the airway surface liquid and therefore mucociliary clearance of respiratory pathogens. A recent study has shown that the E3 ubiquitin ligase Neural precursor cells expressed developmentally downregulated (Nedd4–2) ubiquitinates ΔF508-CFTR in pancreatic epithelial cells and that siRNA-mediated silencing of Nedd4–2 increases plasma membrane ΔF508-CFTR. Because the role of Nedd4–2 in regulating wild-type (wt)-CFTR in airway epithelial cells is unknown, studies were conducted to test the hypothesis that Nedd4–2 also ubiquitinates wt-CFTR and regulates its plasma membrane abundance. We found that Nedd4–2 did not affect wt-CFTR Cl− currents in Xenopus oocytes. Likewise, overexpression of Nedd4–2 in human airway epithelial cells did not alter the amount of ubiquitinated wt-CFTR. siRNA knockdown of Nedd4–2 in human airway epithelial cells had no effect on ubiquitination or apical plasma membrane abundance of wt-CFTR. Thus Nedd4–2 does not ubiquitinate and thereby regulate wt-CFTR in human airway epithelial cells.

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Turnover of the cystic fibrosis transmembrane conductance regulator (CFTR): Slow degradation of wild-type and ΔF508 CFTR in surface membrane preparations of immortalized airway epithelial cells

Journal of Cellular Physiology ◽

10.1002/(sici)1097-4652(199608)168:2<373::aid-jcp16>3.0.co;2-4 ◽

1996 ◽

Vol 168 (2) ◽

pp. 373-384 ◽

Cited By ~ 19

Author(s):

Xiaofang Wei ◽

Robin Eisman ◽

Jin Xu ◽

Alan D. Harsch ◽

Andrew E. Mulberg ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Surface Membrane ◽

Airway Epithelial Cells ◽

Transmembrane Conductance Regulator ◽

Airway Epithelial ◽

Wild Type ◽

Transmembrane Conductance ◽

Δf508 Cftr ◽

Cystic Fibrosis Transmembrane

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Does the ΔF508-CFTR mutation induce a proinflammatory response in human airway epithelial cells?

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00226.2011 ◽

2012 ◽

Vol 303 (6) ◽

pp. L509-L518 ◽

Cited By ~ 15

Author(s):

Thomas H. Hampton ◽

Alicia E. Ballok ◽

Jennifer M. Bomberger ◽

Melanie R. Rutkowski ◽

Roxanna Barnaby ◽

...

Keyword(s):

Epithelial Cells ◽

Inflammatory Response ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Chemokine Production ◽

Human Airway ◽

Δf508 Cftr ◽

Human Airway Epithelial Cells

In the clinical setting, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene enhance the inflammatory response in the lung to Pseudomonas aeruginosa ( P. aeruginosa ) infection. However, studies on human airway epithelial cells in vitro have produced conflicting results regarding the effect of mutations in CFTR on the inflammatory response to P. aeruginosa, and there are no comprehensive studies evaluating the effect of P. aeruginosa on the inflammatory response in airway epithelial cells with the ΔF508/ΔF508 genotype and their matched CF cell line rescued with wild-type (wt)-CFTR. CFBE41o- cells (ΔF508/ΔF508) and CFBE41o- cells complemented with wt-CFTR (CFBE-wt-CFTR) have been used extensively as an experimental model to study CF. Thus the goal of this study was to examine the effect of P. aeruginosa on gene expression and cytokine/chemokine production in this pair of cells. P. aeruginosa elicited a more robust increase in cytokine and chemokine expression (e.g., IL-8, CXCL1, CXCL2 and TNF-α) in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. These results demonstrate that CFBE41o- cells complemented with wt-CFTR mount a more robust inflammatory response to P. aeruginosa than CFBE41o-ΔF508/ΔF508-CFTR cells. Taken together with other published studies, our data demonstrate that there is no compelling evidence to support the view that mutations in CFTR induce a hyperinflammatory response in human airway epithelial cells in vivo . Although the lungs of patients with CF have abundant levels of proinflammatory cytokines and chemokines, because the lung is populated by immune cells and epithelial cells there is no way to know, a priori, whether airway epithelial cells in the CF lung in vivo are hyperinflammatory in response to P. aeruginosa compared with non-CF lung epithelial cells. Thus studies on human airway epithelial cell lines and primary cells in vitro that propose to examine the effect of mutations in CFTR on the inflammatory response to P. aeruginosa have uncertain clinical significance with regard to CF.

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Pseudomonas aeruginosainhibits endocytic recycling of CFTR in polarized human airway epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00108.2005 ◽

2006 ◽

Vol 290 (3) ◽

pp. C862-C872 ◽

Cited By ~ 50

Author(s):

Agnieszka Swiatecka-Urban ◽

Sophie Moreau-Marquis ◽

Daniel P. MacEachran ◽

John P. Connolly ◽

Caitlin R. Stanton ◽

...

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Apical Membrane ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Membrane Expression ◽

Endocytic Recycling ◽

Human Airway ◽

Δf508 Cftr ◽

Human Airway Epithelial Cells

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), ΔF508, leads to the absence of CFTR Cl−channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of ΔF508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl−channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl−secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl−secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and ΔF508-CFTR Cl−channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of ΔF508-CFTR.

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The Short Apical Membrane Half-life of Rescued ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Results from Accelerated Endocytosis of ΔF508-CFTR in Polarized Human Airway Epithelial Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m508944200 ◽

2005 ◽

Vol 280 (44) ◽

pp. 36762-36772 ◽

Cited By ~ 141

Author(s):

Agnieszka Swiatecka-Urban ◽

Andrea Brown ◽

Sophie Moreau-Marquis ◽

Janhavi Renuka ◽

Bonita Coutermarsh ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Half Life ◽

Apical Membrane ◽

Airway Epithelial Cells ◽

Transmembrane Conductance Regulator ◽

Airway Epithelial ◽

Transmembrane Conductance ◽

Δf508 Cftr ◽

Cystic Fibrosis Transmembrane

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The ΔF508-CFTR mutation results in increased biofilm formation byPseudomonas aeruginosaby increasing iron availability

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00391.2007 ◽

2008 ◽

Vol 295 (1) ◽

pp. L25-L37 ◽

Cited By ~ 112

Author(s):

Sophie Moreau-Marquis ◽

Jennifer M. Bomberger ◽

Gregory G. Anderson ◽

Agnieszka Swiatecka-Urban ◽

Siying Ye ◽

...

Keyword(s):

Antibiotic Resistance ◽

Epithelial Cells ◽

Biofilm Formation ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Human Airway ◽

Abiotic Surfaces ◽

Δf508 Cftr ◽

Airway Cells ◽

Human Airway Epithelial Cells

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing ΔF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and ΔF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on ΔF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing ΔF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The ΔF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

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Regulation of endogenous ENaC functional expression by CFTR and ΔF508-CFTR in airway epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00142.2010 ◽

2011 ◽

Vol 300 (1) ◽

pp. L88-L101 ◽

Cited By ~ 39

Author(s):

Ronald C. Rubenstein ◽

Shannon R. Lockwood ◽

Ellen Lide ◽

Rebecca Bauer ◽

Laurence Suaud ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Functional Expression ◽

Surface Expression ◽

Airway Epithelial Cells ◽

Cell Surface Expression ◽

Apical Surface ◽

Airway Epithelial ◽

Airway Epithelia ◽

Δf508 Cftr

The functional expression of the epithelial sodium channel (ENaC) appears elevated in cystic fibrosis (CF) airway epithelia, but the mechanism by which this occurs is not clear. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) alters the trafficking of endogenously expressed human ENaC in the CFBE41o− model of CF bronchial epithelia. Functional expression of ENaC, as defined by amiloride-inhibited short-circuit current ( Isc) in Ussing chambers, was absent under control conditions but present in CFBE41o− parental and ΔF508-CFTR-overexpressing cells after treatment with 1 μM dexamethasone (Dex) for 24 h. The effect of Dex was mimicked by incubation with the glucocorticoid hydrocortisone but not with the mineralocorticoid aldosterone. Application of trypsin to the apical surface to activate uncleaved, “near-silent” ENaC caused an additional increase in amiloride-sensitive Isc in the Dex-treated cells and was without effect in the control cells, suggesting that Dex increased ENaC cell surface expression. In contrast, Dex treatment did not stimulate amiloride-sensitive Isc in CFBE41o− cells that stably express wild-type (wt) CFTR. CFBE41o− wt cells also had reduced expression of α- and γ-ENaC compared with parental and ΔF508-CFTR-overexpressing cells. Furthermore, application of trypsin to the apical surface of Dex-treated CFBE41o− wt cells did not stimulate amiloride-sensitive Isc, suggesting that ENaC remained absent from the surface of these cells even after Dex treatment. We also tested the effect of trafficking-corrected ΔF508-CFTR on ENaC functional expression. Incubation with 1 mM 4-phenylbutyrate synergistically increased Dex-induced ENaC functional expression in ΔF508-CFTR-overexpressing cells. These data support the hypothesis that wt CFTR can regulate the whole cell, functional, and surface expression of endogenous ENaC in airway epithelial cells and that absence of this regulation may foster ENaC hyperactivity in CF airway epithelia.

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Deleterious impact of Pseudomonas aeruginosa on cystic fibrosis transmembrane conductance regulator function and rescue in airway epithelial cells

European Respiratory Journal ◽

10.1183/09031936.00076214 ◽

2015 ◽

Vol 45 (6) ◽

pp. 1590-1602 ◽

Cited By ~ 25

Author(s):

Nguyen Thu Ngan Trinh ◽

Claudia Bilodeau ◽

Émilie Maillé ◽

Manon Ruffin ◽

Marie-Claude Quintal ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Short Circuit ◽

Airway Epithelial Cells ◽

Transmembrane Conductance Regulator ◽

Airway Epithelial ◽

Transmembrane Conductance ◽

Δf508 Cftr ◽

Cystic Fibrosis Transmembrane

The epithelial response to bacterial airway infection, a common feature of lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis, has been extensively studied. However, its impact on cystic fibrosis transmembrane conductance regulator (CFTR) channel function is not clearly defined. Our aims were, therefore, to evaluate the effect of Pseudomonas aeruginosa on CFTR function and expression in non-cystic fibrosis airway epithelial cells, and to investigate its impact on ΔF508-CFTR rescue by the VRT-325 corrector in cystic fibrosis cells.CFTR expression/maturation was evaluated by immunoblotting and its function by short-circuit current measurements.A 24-h exposure to P. aeruginosa diffusible material (PsaDM) reduced CFTR currents as well as total and membrane protein expression of the wildtype (wt) CFTR protein in CFBE-wt cells. In CFBE-ΔF508 cells, PsaDM severely reduced CFTR maturation and current rescue induced by VRT-325. We also confirmed a deleterious impact of PsaDM on wt-CFTR currents in non-cystic fibrosis primary airway cells as well as on the rescue of ΔF508-CFTR function induced by VRT-325 in primary cystic fibrosis cells.These findings show that CFTR function could be impaired in non-cystic fibrosis patients infected by P. aeruginosa. Our data also suggest that CFTR corrector efficiency may be affected by infectious components, which should be taken into account in screening assays of correctors.

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